The present invention relates to methods for identifying nucleic acid molecules encoding (poly)peptides that interact with target molecules. The method of the present invention is particularly characterized by an in vitro translation step under conditions that allow formation of polysomes in the presence of antisense oligonucleotides complementary to the tag-coding sequence of ssrA-RNA. The present invention further relates to kits that are useful for carrying out the method of the invention.
Evolutionary methods may bring the refinement to protein engineering which is beyond the powers and accuracy of rational design today. Evolution can be defined as a succession of xe2x80x9cgenerationsxe2x80x9d, cycles of genetic diversification, followed by Darwinian selection for a desired phenotypic property. In classic experiments, nucleic acids have been evolved for physical properties (Saffhill, R., Schneider-Bernloehr, H., Orgel, L. E. and Spiegelman, S. (1970) J. Mol. Biol. 51, 531-539) in vitro, and in this case, the substance conferring the phenotype was identical to the genetic material. Oligonucleotide ligands, usually single stranded RNA, have been identified for many targets by SELEX (Gold, L., Polisky, B., Uhlenbeck, O. and Yarus, M. (1995) Annu. Rev. Biochem. 64, 763-797; Irvine, D., Tuerk, C. and Gold, L. (1991) J. Mol. Biol. 222, 739-761), in which a synthetic DNA library is transcribed, the RNA selected for binding, reverse transcribed and amplified over several rounds. Early experiments with proteins as the carrier of the phenotype, clearly of much broader applicability, had relied on living cells for effecting the coupling between gene and protein, either directly or via the production of phages or viruses (Phizicky, E. M. and Fields, S. (1995) Microbiol. Rev. 59, 94-123). Since in this type of experiment the DNA library as the information carrier for encoded protein diversity has to be transformed or transfected into bacterial or eukaryotic cells, the available diversity was severely limited by the low efficiency of DNA uptake (Dower, W. J. and Cwirla, S. E. (1992) in Guide to Electroporation and Electrofusion, eds. Chang, D. C., Chassy, B. M., Saunders, J. A. and Sowers, A. E. (Academic Press, San Diego), pp. 291-301). Furthermore, in each generation, the DNA library had to be first ligated into a replicable genetic package by which diversity was again decreased. In addition, many promising variants would have to be selected against in the host environment. Only very few studies (Yang, W. P., Green, K., Pinz-Sweeney, S., Briones, A. T., Burton, D. R. and Barbas 3rd., C. F. (1995) J. Mol. Biol. 254, 392-403) have carried protein optimization through more than one generation using methods such as phage display, since this requires repeated switching between in vitro diversification and in vivo screeningxe2x80x94a laborious process.
With the goal of circumventing or improving this process, a number of laboratories have designed novel systems that are based on the immediate vicinity and physical connection of mRNA and corresponding (poly)peptides during translation. Thus, a series of studies have shown that specific mRNAs can be enriched by immunoprecipitation of polysomes (Schechter, I. (1973) Proc. Natl. Acad. Sci. U.S.A. 70, 2256-2260; Payvar, F. and Schimke, R. T. (1979) Eur. J. Biochem. 101, 271-282; Kraus, J. P. and Rosenberg, L. E. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 4015-4019). Recently, Mattheakis and coworkers reported an affinity selection of a short peptide from a library using polysomes, in order to connect genotype and phenotype in vitro (Mattheakis, L. C., Bhatt, R. R. and Dower, W. J. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 9022-9026; W095/11922).
This system employs an in vitro translation system that is preferentially coupled to an in vitro transcription system. The translation system allows the simultaneous isolation of mRNA and (poly)peptide in a polysome complex after a suitable screening step for the (poly)peptide. Preferably, the (poly)peptides in Mattheakis"" system are comprised of two components, one of which is the peptide to be screened and the second is a tether segment that binds to the mRNA. Co-isolation of mRNA and (poly)peptide in the polysome complex can possibly be improved with the help of a translation stalling sequence even though the existence of such sequences is still unclear for E. coli. This sequence possibly enhances the overall stability of the polysome complex by decreasing the translation rate and thus allows for suitable conditions for the concomitant screening and isolation of (poly)peptide and corresponding mRNA.
Similar work has earlier been reported by Gold and colleagues (WO 93/03172) and Kawasaki and coworkers (WO 91/05058). Although the above-described systems have established a means of characterizing a nucleic acid via the identification of a protein encoded by said nucleic acid, there are practical limitations with respect to the efficiency of the ribosome displays of the nascent (poly)peptide. The technical problem underlying the present invention was therefore to increase the efficiency of (i) synthesis of a collection of stable RNA molecules and (ii) translation of said RNA molecules, and thereby to achieve an increased efficiency of the use of polysomes in screening. The solution to said technical problem is achieved by providing the embodiments characterized in the claims.
Accordingly, the present invention relates to a method for identifying a nucleic acid molecule encoding a (poly)peptide that interacts with a target molecule comprising the following steps:
(a) translating a population of mRNA molecules devoid of stop codons in the correct reading frame in an in vitro translation system, said translation system either comprising antisense oligonucleotides complementary to the tag-coding sequence of ssrA-RNA or being free of ssrA-RNA, under conditions that allow the formation of polysomes;
(b) bringing the polysomes so formed into contact with said target molecules under conditions that allow the interaction of the (poly)peptides encoded by said mRNA molecules and displayed by said polysomes with said target molecules;
(c) separating polysomes displaying (poly)peptides that interact with said target molecules from polysomes displaying no such (poly)peptides; and
(d) identifying the nucleic acid molecule encoding a (poly)peptide displayed in a polysome that interacts with said target molecules.
The term xe2x80x9c(poly)peptidexe2x80x9d as used in the present invention relates both to peptides as well as to polypeptides. Said (poly)peptides may either comprise a natural or a recombinantly engineered amino acid sequence. The latter alternative also includes fusion proteins.
According to the present invention, the term xe2x80x9cpolysomexe2x80x9d refers to a complex formed by at least one, preferably several ribosomes and mRNA during translation.
The population of mRNA molecules may be of varying origin. For example, it may be derived from a cDNA library. In an alternative embodiment, it may be directly derived from cells or tissue. Particularly advantageous is also the use of the present invention in mutagenized (poly)peptides to find improved variants. Alternatively, synthetic protein or peptide libraries or antibody libraries can be used. The term xe2x80x9ctag-coding sequence of ssrA-RNAxe2x80x9d relates to a nucleic acid sequence encoding the amino acid sequence AANDENYALAA (SEQ ID NO;1). This sequence has been described in Keiler et al., Science 221 (1996), 1990-1993.
The antisense oligonucleotides comprised in the translation system employed in the method of the invention are of a suitable length to hybridize to the tag-coding sequence of ssrA-RNA and block translation thereof under conditions that allow the formation of polysomes.
A translation system being free of ssrA-RNA can, for example, be derived from E. coli strains lacking a functional ssrA gene such as X90 ssrA1::cat (Keiler et al., Science 221 (1996), 1990-1993), N2211 or NM101 (Tu et al., J. Biol. Chem. 270 (1995), 9322-9326), W3110 xcex94ssrA (Komine et al., Proc. Natl. Acad. Sci. U.S.A. 91 (1994), 9223-9227), K7823 or K6676 (Retallack and Friedman, Cell 83 (1995), 227-235).
Conditions that allow the interaction of the (poly)peptides encoded by the translated mRNA molecules and displayed by said polysomes with corresponding target molecules can, without undue burden, be established by the person skilled in the art. Such conditions are, for example, derivable from the teachings of WO 95/11922, WO 93/03172 and WO 91/05058 or from the appended examples. As is well known in the art, said conditions also rely on the screening procedure that is employed for detecting said interactions.
The separation of the polysomes that display (poly)peptides which interact with the target molecules from polysomes displaying no such (poly)peptides can be effected according to known procedures. Again, the separation technique employed may well depend on the screening system that is used. A convenient method of separating the aforementioned polysomes is, for example, based on affinity chromatography wherein the target molecules are bound to the column material.
The identification of the nucleic acid molecule encoding the selected (poly)peptide can be achieved by any suitable means and is most conveniently achieved by sequencing the nucleic acid molecule, for example, by sequencing the mRNA or the DNA, after cloning into a vector. For identification of the mRNA, it may be removed from the ribosome by treatment with EDTA or by acid elution followed by standard RNA purification using a kit (see Example 3) or by competitive elution using a soluble target molecule, followed by standard RNA purification using a kit.
Preferably and most advantageously, steps (a) to (c) are carried out two or more times prior to the identification step (d). This measure results in the less ambiguous identification of the desired nucleic acid with the concomitant minimization of false positive polysomes and thus nucleic acids. This embodiment for identifying the desired nucleic acid is particularly preferred, if several rounds of selection are necessary to isolate the specifically interacting (poly)peptide-target molecule pair.
In accordance with the present invention, it has surprisingly been found that by including antisense oligonucleotides complementary to the tag-coding sequence of ssrA-RNA results in a manifold increase in the efficiency of polysome display. This result is all the more unexpected since the prior art referred to above had already tried by various routes and means to establish such optimal conditions.
The present invention therefore provides a system for the phenotypic selection of target molecules such a ligands with (poly)peptides that are preferably complete, native protein molecules.
In a preferred embodiment of the present invention, the mRNA molecules comprise a stem loop at their 3xe2x80x2 end.
In this embodiment of the invention, degradation of the mRNA by exonucleases is precluded to a significant extent.
Most preferably, a spacer is fused to the reading frame of the (poly)peptide to tether the emerging, folded (poly)peptide to the putative (poly)peptide channel of the ribosome. Said spacer preferentially encodes 57 to 116 amino acids.
Tethering of emerging (poly)peptide to the (poly)peptide channel of the ribosome is an additional advantageous means to enable co-selection of (poly)peptide and corresponding mRNA since it might significantly slow down the dissociation of the polysome.
In a further most preferred embodiment said stem loop region at the 3xe2x80x2 end of the mRNA molecules encodes said spacer. Thereby, the length of the total 3xe2x80x2 region can be kept to a minimum, if both spacer (required at the protein level) and stem loop (required at the RNA level) can be encoded by the same DNA.
A further preferred embodiment of the present invention relates to the above-recited method wherein said mRNA molecules comprise a stem loop structure at their 5xe2x80x2 end.
Like the stem loop structure at the 3xe2x80x2 end, the stem loop at the 5xe2x80x2 end of the mRNA serves the purpose of avoiding a successful exonuclease attack towards the mRNA. It is particularly preferred that the mRNA comprises both a 5xe2x80x2 and a 3xe2x80x2 stem loop structure. In this embodiment, the mRNA molecule would structurally resemble natural mRNAs.
In an additional preferred embodiment of the method of the invention, said in vitro translation system is supplemented with inhibitors of ribonuclease.
Preferably, said ribonuclease inhibitors are transition state analogs and most preferably they are vanadyl ribonuclease complexes.
In accordance with the present invention, it was found that in particular vanadyl ribonuclease complexes may advantageously be used to further increase the efficiency of ribosome display. This result is particularly surprising since said complexes at the same time partially inhibit protein synthesis.
A further preferred embodiment of the invention relates to a method wherein the polysomes in steps (a) to (c) are stabilized by
(a) the addition of magnesium salts, preferably magnesium acetate, after the formation of polysomes; and/or
(b) a means that forms a bridge between the mRNA and the corresponding (poly)peptide; and/or
(c) a low temperature after the translation and/or during the screening process.
The above recited means have been proven to further enhance the stability of the polysomes. Thus, a 50 mM magnesium acetate concentration in the reaction buffer significantly stabilized the ribosome complexes against dissociation. The term xe2x80x9clow temperaturexe2x80x9d in the above context is intended to mean a temperature that allows a successful screening to take place. Preferably said low temperature is in the range of 0 to 50xc2x0 C.
Preferably, the translation is carried out in a prokaryotic translation system. Particularly preferred is an E. coli based translation system such as the S-30 E. coli translation system.
Alternatively, the translation system may be carried out in a eukaryotic translation system.
In a further preferred embodiment of the method of the present invention step (d) comprises
(da) reverse transcribing said mRNA;
(db) optionally amplifying the resulting cDNA;
(dc) optionally cloning the optionally amplified cDNA; and
(dd) determining the sequence of said cDNA.
This embodiment for identifying the nucleic acid of interest is preferred, if the population of mRNA molecules is too large to identify the desired species in a single round. Furthermore, it allows repeated and detailed testing of identified molecules, since the population of mRNA molecules becomes xe2x80x9cimmortalizedxe2x80x9d by cloning.
Reverse transcription allows sequencing using the most convenient DNA sequencing technology developed by Maxam and Gilbert as well as by Sanger and colleagues (see, e.g., Sambrook et al., xe2x80x9cMolecular Cloning, A Laboratory Manualxe2x80x9d, second edition 1989, CSH Press, Cold Spring Harbor).
The amplification of cDNA, preferably by PCR, with or without subsequent cloning into a suitable vector, further significantly facilitates the identification of the desired nucleic acid molecule. In various cases, amplification of the nucleic acid molecule will be a prerequisite for allowing the investigator to subsequently identify said nucleic acid molecule.
In an additional preferred embodiment of the method of the invention, DNA is transcribed into mRNA in the presence of a reducing agent, such as b-xcex2-mercaptoethanol and/or DTT, prior to step (a).
The inclusion of a reducing agent such as xcex2-mercaptoethanol and/or DTT into the reaction buffer is known to increase the stability of DNA-polymerase. Accordingly, a reducing agent in the buffer contributes to an increase in the yield of mRNA which, in turn, results in an overall improvement of the ribosome display.
It is particularly preferred to remove said reducing agent after transcription and prior to step (a). This method of the invention is most preferred in cases where the (poly)peptide to be screened may comprise species that assume their native conformation by forming disulfide bridges. An example of such (poly)peptides are members of the immunoglobulin superfamily. By introducing this preferred embodiment, the present invention directly contradicts the prior art referred to above that suggests using a combined transcription/translation system.
Furthermore, the present invention also relates to a method for identifying a nucleic acid molecule encoding a (poly)peptide that interacts with a target molecule comprising the following steps:
(a) transcribing a population of DNA molecules devoid of stop codons in the correct reading frame into the corresponding population of mRNA molecules in the presence of a reducing agent;
(b) removing said reducing agent from said population of mRNA molecules;
(c) translating said population of mRNA molecules in an in vitro translation system under conditions that allow the formation of polysomes;
(d) bringing the polysomes so formed into contact with said target molecules under conditions that allow the interaction of the (poly)peptides encoded by said mRNA molecules and displayed by said polysomes with said target molecules;
(e) separating polysomes displaying (poly)peptides that interact with said target molecules from polysomes displaying no such (poly)peptides; and
(f) identifying the nucleic acid molecule encoding a (poly)peptide displayed in a polysome that interacts with said target molecules.
In a preferred embodiment the reducing agent used in step (a) is xcex2-mercaptoethanol and/or DTT.
In a further preferred embodiment of the method of the present invention, the (poly)peptides comprise domains of the immunoglobulin superfamily, and preferably of the immunoglobulin family.
For example, said (poly)peptides may comprise complete T cell receptor or antibody chains or parts thereof such as domains of antibodies, for example the VH or VL regions.
It is particularly preferred that the (poly)peptides are single chain antibodies or fusion proteins comprising such single chain antibodies. In the latter alternative, the fusion partner of said antibody chains preferably is a tag that is employed for tethering the nascent (poly)peptide to the corresponding mRNA.
The present invention also preferably relates to a method wherein the translation system is supplemented with at least one compound selected from the group consisting of protein disulfide isomerase, oxidized or reduced glutathione, E. coli protein DsbA and molecular chaperones such as DnaK, DnaJ, GrpE, GroEL or GroES.
The above compounds, alone or in combination, may enhance the stability, solubility and/or native folding capacities of the nascent (poly)peptide.
The protein disulfide isomerase may be of bacterial or eukaryotic origin. The compound/enzyme that is included into the system would be selected by the person skilled in the art according to the type of protein that is screened. For example, if he screens a library comprising antibody domains he would, in accordance with the teachings of the present invention, include a eukaryotic protein disulfide isomerase. As could be shown by the present invention (see the appended examples) the polysome display system is significantly improved by incorporating said enzyme into the translation reaction system.
In a further preferred embodiment of the method of the present invention, non-specific interactions between the polysomes and/or the polysomes and the target molecules and/or, optionally, the polysomes and the matrix on which the target molecules are immobilized, formed during the step of bringing the polysomes into contact with said target molecules are inhibited or reduced by the addition of a blocking compound.
In a most preferred embodiment, said blocking compound is a polyanionic compound like heparin.
Heparin has been suggested to be included as RNase inhibitor (WO 91/05058), but it has surprisingly been found in accordance with the present invention that it additionally decreases non-specific binding. It can be assumed that heparin as polyanionic compound competes with the polyanionic mRNA as part of the polysome complexes for non-specific binding sites, rendering the addition of polyanionic compounds such as heparin in polysome display a generally applicable method for decreasing non-specific binding.
In another most preferred embodiment, said blocking compound is sterilized milk. The addition of nonfat milk has already been suggested for polysome display (WO 95/11922). However, according to the present invention it has been found that no RNA could be isolated when milk was used during affinity selection. Surprisingly, when sterilized milk was used, RNA isolation was again possible, and the amount of non-specific binding was substantially decreased.
Furthermore, the present invention relates to a kit comprising
(a) antisense oligonucleotides complementary to the tag-coding sequence of ssrA-RNA;
(b) optionally a vector suitable for cloning nucleic acids encoding (poly)peptides to be screened;
(c) optionally, ribonuclease inhibitors, preferably transition state analogs, and most preferably vanadyl ribonucleoside complexes;
(d) optionally, at least one compound selected from the group consisting of a protein disulfide isomerase, oxidized or reduced glutathione, E. coli DsbA, and molecular chaperones; and
(e) optionally oligonucleotides encoding 5xe2x80x2 or 3xe2x80x2 stem loops, spacers or terminators without stop codons.
In a preferred embodiment the kit according to the invention may furthermore comprise:
(f) S-30 translation extract;
(g) PCR components;
(h) reverse transcriptase;
(i) an RNA sequencing kit;
(j) a DNA sequencing kit, either alone or in combination.
Finally, the present invention relates to a kit comprising
(a) an in vitro cell-free translation extract free of ssrA-RNA;
(b) optionally a vector suitable for cloning nucleic acids encoding (poly)peptides to be screened;
(c) optionally, ribonuclease inhibitors, preferably transition state analogs, and most preferably vanadyl ribonucleoside complexes;
(d) optionally, at least one compound selected from the group consisting of a protein disulfide isomerase, oxidized or reduced glutathione, E. coli DsbA, and molecular chaperones; and
(e) optionally oligonucleotides encoding 5xe2x80x2 or 3xe2x80x2 stem loops, spacers or terminators without stop codons.
In a preferred embodiment the kit according to the invention may furthermore comprise:
(f) PCR components;
(g) reverse transcriptase;
(h) an RNA sequencing kit;
(i) a DNA sequencing kit, either alone or in combination.
The kit of the present invention can conveniently be used to carry out the method of the present invention.